Chemically modified silicas have been, and continue to be, widely used as supports in a great variety of chromatographic separations. With the aim of controlling its selectivity while reducing unwanted interactions with one or more compounds, numerous synthetic procedures have been developed to attach organic moieties (R) on the silica surface. Early work on the chemical modification of silica described the use of an esterification reaction between surface silanol groups and an alcohol to give Si-O-C linkages (Halasz and Sebastian, Angew. Chem. (Int. Ed.) 8:453 (1969); Deuel, et al., HeIv. Chim. Acta 119:1160 (1959)). Although such materials were useful for many separations, their limited hydrolytic stability seriously precluded the extensive usage of these bonded phases, particularly in liquid chromatography which requires the use of aqueous eluents.
Currently, commercially available bonded phases are prepared by reacting selected organosilanes with the silica surface. Halogen- or alkoxy-substituted alkyldimethylsilanes react with the surface silanols attached to the silica surface through an Si-0-Si-C linkage. By using this approach it is possible to produce bonded silicas with a great variety of organic groups, ranging from non-polar materials, for instance, octyl- and octadecyl-silicas (R --(CH.sub.2).sub.n CH.sub.3, with n-7 and 17 respectively) commonly used as bonded supports in reversed-phase liquid chromatography, to ionic materials such as benzenesulphonic acid derivatives, which are widely used in ion-exchange liquid chromatography. The preparation of these and similar materials are described in a number of publications (e.g., Roumeliotis and Unger, J. Chromatogr. 149:211 (1978) or Asmus, et al., J. Chromatogr. 123:109 (1976)) and patents (Sebastian, et al., U.S. Pat. No. 3,956,179; Hancok, et al., U.S. Pat. No. 4,257,916; or Ramsden, et al., U.S. Pat. No. 4,661,248).
The new development of electrophoretic separations in a capillary format has promoted the extent of the silane technology normally used in chromatography to the deactivation of the inner wall of the quartz capillary. Thus, Jorgenson, et al. (Science 222:266 (1983)) have noted that separation of model proteins, such as cytochrome, lysozyme and ribonuclease A, in untreated fused silica capillaries with a phosphate buffer at pH 7 was accompanied by strong tailing, and suggested that this might be caused by strong interactions of the proteins and the capillary wall. Derivatization of the capillary wall has been proven effective to prevent or control protein sorption. In another application (U.S. Pat. No. 4,680,201 issued 1987), Hjerten describes a method for preparing a thinwall, capillary tube for electrophoretic separations by use of a bifunctional compound in which one group (usually a terminal --SiX.sub.3 group where X=Ethoxy, methoxy or chloride) reacts with the glass wall and the other (usually an olefin group) does so with a monomer taking part in a polymerization process. This process resulted in a wall-bonded, polymer-filled capillary useful for polyacrylamide gel electrophoresis. In addition, by chemically modifying the quartz surface of the capillary, operational variables such as the electroosmotic flow are said to be more amenable to control.
The extensive usage of these bonded materials in chromatography and capillary electrophoresis does not necessarily imply that they meet all requirements with respect to separation performance and stability. On the contrary, they are subject to serious effects arising primarily from a relatively limited organic coverage due to the "bulky" methyl groups of the silane reagent and from a still unsatisfactory hydrolytic stability, particularly under moderately acidic or slightly alkaline elution conditions. This limited surface coverage along with a hydrolytically labile organic layer both result in the exposure of a substantial number of surface silanols, groups which are known to be primarily responsible for the residual adsorption phenomena that plague silicon-based materials. These so called "silanophilic" interactions are usually undesirable in chromatography as well as in capillary electrophoresis because they often result in "tailing" peaks, catalyzed solute decomposition, lead to unreliable quantitation, etc. One of the most striking cases of silanophilic interactions occurs perhaps in the separation of certain compounds containing amino or other similar groups, particularly biomolecules. For instance, many proteins may interact very strongly with unreacted silanols leading to excessive band tailing, incomplete recovery of one or more solutes, or even recovery of the same component from different bands. As a result of such problems, other organosilane reagents have been developed.
Two related approaches have been proposed in which the methyl groups of the organosilane reagent are replaced either by a "bidentate" or by a bulkier group (Kirkland and McCormick, Chromatographia 24:58 (1987)). In both cases the new groups are aimed to shield the unreacted silanols as well as the hydrolytically labile linkage that bonds the silane to the support. Although this steric protection has resulted in somewhat improved bonded phases, the necessity still exists for a truly effective silane chemistry.
In another completely different approach, bonded silicas bearing direct Si-C linkages have been developed. They involve the sequential reaction of the silica substrate with a chlorinating reagent (e.g., thionyl chloride) and a proper alkylating reagent (e.g., a Grignard or organolithium compound). In principle this method should provide not only a closer attachment and a denser coverage of organic functionalities but also a more hydrolytically stable bonded phase than that obtained by the corresponding Si-O-Si-C linkage. Nevertheless, the application of chlorination/Grignard or chlorination/organolithium reaction sequence to modify silica substrates has been hindered by several factors. One factor is that the one-step organosilanization (such as described in U.S. Pat. No. 3,956,179 to Sebastian, et al.) is relatively easy to produce materials as compared to the two-step halogenation/alkylation which, additionally, demands rigorous moisture-free conditions. Difficulties associated with the removal of residual salts which may be occluded in the porous silica matrix during the alkylation process is also an important factor which has contributed to the limited usage of this synthetic approach. Additionally, the preparation of the alkylation reagent exhibits strong interferences with many reactive functionalities, particularly those containing carbonyl, nitrile, carboxyl, amide, alcohol, etc. That is, the great reactivity which makes a Grignard reagent so useful in many synthetic approaches seriously limits its applicability. The organic group, R, in the Grignard reagent, RMgBr, must remain intact during the preparation of the reagent. It is a well known fact that Grignard reagents react with acidic components to form the corresponding hydrocarbon group R-H. More strictly, "any compound containing hydrogen attached to an electronegative element such as oxygen, nitrogen, and even triply-bonded carbon are acidic enough to decompose a Grignard reagent" (Morrison and Boyd, Organic Chemistry, 3rd Edition, 1974). Additionally, a Grignard reagent reacts readily with molecular oxygen, carbon dioxide, and with " nearly every organic compound containing a carbon-oxygen or carbon
nitrogen multiple bond" (supra). The group nitro (--NO.sub.2) also appears to react oxidatively with a Grignard reagent. It seems clear therefore that only a very limited number of organic functionalities may be present in the halide compound from which a Grignard reagent can be prepared. Being even more reactive than the corresponding Grignard reagent, an organolithium reagent should exhibit the same limitations described above in a similar or greater extent. This, of course, greatly limits the versatility of this approach.
It is therefore desirable to address the shortcomings of existing bonded packings by developing an alternate silane chemistry which combines the superior coverage and hydrolytic stability of direct Si-C linkages with the preparation simplicity of silane derivatization.